Method and devices for analyzing small RNA molecules

Abstract

The instant inventon provides methods and devices for detecting, enumerating and / or identifying small RNA molecules using single molecule sequencing techniques.

Description

TECHNICAL FIELD OF THE INVENTION[0001]This invention relates to methods, devices, and combination articles of manufacture for detecting, enumerating, and identifying small RNAs. According to the invention, small RNAs are modified with an adaptor such that they can be attached to a surface for sequence analysis.BACKGROUND OF THE INVENTION[0002]Small RNAs are repressors of gene expression found ubiquitously in eukaryotes. Small RNAs are typically about 21 to about 26 nucleotides in length and induce repression through homologous sequence interactions. There are many types of small RNAs including short interfering (si)RNAs, small temporal (st)RNAs, heterochromatic siRNAs, tiny non-coding RNAs, and micro (mi)RNAs. Small RNAs can control mRNA stability or translation, or target epigenetic modifications to specific regions of the genome. Small RNAs are typically produced by processing of longer double-stranded RNA precursors by an RNaseIII-like enzyme.[0003]Small RNAs regulate gene expres...

AI Technical Summary

Benefits of technology

[0007]The invention provides methods, apparatus, and compositions for the detection, enumeration, and identification of small RNA molecules. According to methods of the invention, detection of small RNA molecules is achieved by attaching the modified small RNA to a surface at single molecule resolution, and analyzing the sequence of the attached small RNA molecules. The invention provides sample preparation, attachment strategies, surface preparation, and rinsing strategies that result in improved detection, enumeration, and identification of small RNA molecules in a biological sample.

[0012]According to one embodiment of the invention, the surface comprises a layer of epoxide molecules arranged in a substantially uniform way, for example, substantially in the form of a monolayer. In some embodiments, which may include any of the elements described below, it is advantageous to block non-specific binding sites that may interfere with detection of incorporation events during nucleic acid sequencing reactions. Agents such as water, sulfate, an amine group, a phosphate or a detergent may be used to block non-specific binding. A detergent, such as Tris, can serve to block or passivate the epoxide molecules alone or in conjunction with other blocking agents. Thus, a detergent may be incorporated into surface washing steps in order to preserve a passivated surface and prevent excess background that may interfere with detection. Blocking can occur by exposing the surface to molecules that compete with non-specific binding or that reduce or eliminate the reactive portion of the surface molecule. For example, water can open the epoxide ring, making it less reactive. Thus, after attachment of primers or small RNA molecules, an epoxide surface can be rinsed in order to reduce or eliminate the reactive functionality of the epoxide, thus reducing non-specific binding.

Problems solved by technology

However, computational analyses of genomes have revealed that many more miRNAs are likely to exist that have eluded the various cloning strategies to date.

Detection by Northern blotting is problematic because of the low sensitivity of the assay, often requiring microgram quantities of RNA.

In addition, the transfer required by Northern blotting often has low reproducibility of RNA to a solid support, required by Northern blotting due to the small size of the RNA target molecules.

RNase protection assays are less desirable because of the requirement for highly radioactive probes.

Cloning of individual small RNAs followed by sequencing is effective in determining single-base differences between closely related small RNAs, however the technique is time consuming and thus far not amenable to high throughput.